The earliest known step in mammalian development is the formation of the trophoblast from the totipotential embryonic stem cell. The subsequent differentiation of the trophoblast stem cells (TS) to the placental syncytiotrophoblast (SynT) establishes the epithelial interface between fetal and maternal circulatory systems, mediating essential nutrient exchange and oxygen transport. The SynT also serves as the primary site of fetal hormone production that conditions maternal and fetal systems to sustain a successful gestation. The major hormones synthesized in the SynT are chorionic somatomammotropin (hCS) and growth hormone-variant (hGH-V). Both hormones are encoded in the multi-gene human growth hormone (hGH) gene cluster that also encompasses the pituitary growth hormone gene, hGH-N. We hypothesize that the selective and robust activation of the placental hCS and hGH-V genes during differentiation of TS to SynT reflects a defined progression of epigenetic modifications and alterations in chromatin structures that distinguish this pathway from that involved in activation of hGH-N in pituitary somatotropes. We further hypothesize that this developmental progression reflects global epigenetic controls that are fundamental to SynT differentiation and distinguish this lineage from the TS-derived lineage that leads to the formation of invasive trophoblast cells. The selective activation of the placentally-expressed genes from the hGH cluster thus presents an optimal model for understanding mechanisms of gene activation that underlie TS cell differentiation and that define function(s) of SynT cells in the placenta. Our proposal will address five Specific Aims: I. Establish mouse TS lines that model activation of the hGH locus during SynT differentiation. II. Define the structural alterations at the hGH locus that coincide with SynT-lineage commitment and the subsequent transcriptional induction of the hCS/hGH-V genes. III. Establish mechanistic linkages between SynT differentiation and hGH locus activation. IV. Identify structural features of the hGH locus required for activation in SynT. V. Define higher-order conformations at the hGH chromatin locus in SynT necessary for placenta-specific gene activation. These studies should expand our understanding of placental gene expression and development and expand our insights into the corresponding defects that impact on maternal and fetal health during gestation.